Direct Amplification-Free Nanopore Sequencing of Leishmania from Cutaneous Biopsies

Background: PCR confirms cutaneous leishmaniasis but yields limited species data. Direct nanopore sequencing could offer broader genomic evidence without amplification, though the value of long-read sequencing depends on target recovery, accurate species assignment, and cost. Methods: Sixteen biopsy-DNA extracts (15 PCR-positive, one negative) were sequenced without target amplification using 16-plex adaptive human depletion. Three extracts were additionally analyzed via standard sequencing and adaptive target enrichment. A positive call required at least three nuclear Leishmania reads spanning distinct 10-kb loci across at least two chromosomes. Species evidence was evaluated by competitive alignment, Kraken2, and markers. Results: Fourteen PCR-positive biopsies met the detection rule (93.3% agreement) within 3.5 h of sequencing with the negative biopsy yielding no qualifying reads. Adaptive enrichment increased the target fraction 3.1-5.8-fold but recovered only 12-62% of the absolute target bases obtained by standard sequencing and did not shorten detection time. Unexpected L. aethiopica assignments motivated a held-out benchmark, revealing that classifiers systematically misassigned 97.1-98.3% of L. tropica fragments to L. aethiopica due to reference bias. Marker analysis provided a sparse but useful locus-specific check contrasting these assignments. Modeled sequencing costs (US$30-72/specimen) exceeded endpoint PCR (US$5). Conclusions: PCR-free nanopore sequencing recovers conservative multi-locus L. eishmania evidence directly from biopsies. Adaptive enrichment improved target fraction without improving absolute recovery, while incomplete reference representation limited species interpretation even when genome-wide classifiers agreed. The current approach is therefore better positioned as a genomic adjunct for surveillance settings than as a replacement for routine PCR.